Epigenetic regulations, including chromatin accessibility, nucleosome positioning, and DNA methylation intricately shape genome function. However, current chromatin profiling techniques relying on short-read sequencing technologies fail to characterise highly repetitive genomic regions and cannot detect multiple chromatin features simultaneously. Here, we performed Simultaneous Accessibility and DNA Methylation Sequencing (SAM- seq) of purified plant nuclei. Thanks to the use of long-read nanopore sequencing, SAM-seq enables high-resolution profiling of m6A-tagged chromatin accessibility together with endogenous cytosine methylation in plants. Analysis of naked genomic DNA revealed significant sequence preference biases of m6A-MTases, controllable through a normalisation step. By applying SAM-seq to Arabidopsis and maize nuclei we obtained fine-grained accessibility and DNA methylation landscapes genome-wide. In addition, we uncovered crosstalk between chromatin accessibility and DNA methylation within nucleosomes of genes, TEs, and centromeric repeats. SAM-seq also facilitated the identification of DNA footprints over cis-regulatory regions. Furthermore, using the single-molecule information provided by SAM-seq we identified extensive cellular heterogeneity at chromatin domains harbouring antagonistic chromatin marks, suggesting that bivalency reflects cell-specific regulations of gene activity. SAM-seq is a powerful approach to simultaneously study multiple epigenetic features over unique and repetitive sequences, opening new opportunities for the investigation of epigenetic mechanisms in model as well non-model plant species with limited genomic and epigenomic information.

SAM-Seq was performed on seven day old leaves of maize accession B73. Bigwig file of mCG, mCHG, mCHH methylation and accessibility over the AGPv4 reference genome.